Prostate cancer progression can be influenced 1-levels of androgens and estrogens which regulate cell growth and gene expression, 2-presence of immune cell infiltrates and cytokine signaling, and 3-the stromal tissue microenvironment which can regulate epithelial normal or cancer cell function. The NCCAM Intramural Endocrine laboratory has developed an endocrine-immune-paracrine laboratory model to study the effects of selected androgenic (dehydroepiandrosterone (DHEA)), estrogenic (phytoestrogens) or other dietary supplements and/or natural products on neoplastic prostate epithelial and stromal cell growth, gene expression, including signaling between stromal and epithelial cells and the intracellular signal transduction pathways. DHEA is produced by the adrenal cortex and is the most abundant steroid in humans and can be metabolized in the tissues to androgens or estrogens. Serum levels of DHEA and DHEAS peak in men and women in the 3rd decade and decrease progressively and profoundly with age. In the U.S., DHEA is widely available as an over-the-counter dietary supplement, and is increasingly self-prescribed for its alleged anabolic and anti-aging effects, etc., but its safety and efficacy remain uncertain. As a precursor to both estrogen and testosterone, DHEA excess may pose a potential cancer risk in hormone-responsive tissues such as the prostate. We have investigated effects of DHEA on epithelial and stromal cells from normal and cancer prostate tissues, grown separately and in co-culture. Monocultures of epithelial or stromal cells may miss crucial mechanisms of DHEA effects. Important mechanisms of DHEA effects on prostate cells were discovered when cancer cells were grown in the presence of stromal cells, which showed increased stromal testosterone production and epithelial PSA protein expression. It has been suggested that about 20% of human cancers are associated with chronic infection or inflammation. Such lesions have been characterized in the prostate as proliferative inflammatory atrophy (PIA) and illustrate the association between inflammation and unusually high proliferation. TGF beta-1 was previously shown to induce an immuno-reactive prostatic stroma as is associated with the cancer tissue microenvironment. In these studies, TGF beta-1 was added to stromal cells inducing the reactive stroma phenotype and also produced a marked increase in PSA protein secretion and gene expression in cocultured epithelial cells. Also in cocultures of LAPC-4 + 6S cells, TGF beta-1 greatly increased metabolism of DHEA to testosterone. These results raise a provocative hypothesis that in cancer tissues, compared to normal prostate, the presence of inflammatory factors may induce metabolism of DHEA to androgenic ligands, and may increase induction of prostate epithelial growth and PSA production. In the inflammatory prostate tissue microenvironment, such as in PIA or prostate intraepithelial neoplasia (PIN), is it possible that one of the mechanisms of cancer promotion includes increased metabolism of endogenous DHEA, either to androgens or estrogens, and also increased induction of paracrine factors (including cytokines, chemokines and growth factors), that induce proliferation or inhibit apoptosis. The coculture model of endocrine-immune-paracrine interactions in the prostate, developed in the laboratory has translational applications providing a tool for discovery of natural products or traditional medicines that may be involved in stromal-epithelial cell interactions especially altering paracrine hormonal signals. (see Gray et al, 2008) For instance, red clover contains various isoflavones, including genistein, daidzein biochanin A, and formononetin. In prostate LAPC-4/6S cocultures, red clover isoflavones decreased PSA protein and gene expression and T metabolism induced by TGF beta-1 +DHEA in a dose dependent manner. Prostate cancer (PrCa) risk is positively associated with levels of insulin-like growth factor I (IGF-I) and PSA, both of which are androgen receptor (AR) signaling target genes in PrCa cells. IGF-1 is an important paracrine signal between stromal and epithelial cells. We found that IGF-1 is required, as both a paracrine and an autocrine factor for R1881-induction of epithelial PSA expression, and functional depletion of IGF-I diminished the induction of PSA. This pathway also involved accumulation of cytoplasmic dephospho beta-catenin (CPDP beta -catenin) and phosphoinositide 3-kinase (PI3K) since inhibiting this kinase activity abolished R1881-induced Akt phosphorylation, and accumulation of CPDP and nuclear beta-catenin, resulting in abrogating R1881-induced expression of IGF-I and/or PSA (Liu,2010 in revision). We also reported non-androgenic mechanisms of DHEA in androgen independent prostate cancer cells. DHEA induced beta-Catenin/TCF signaling (beta-CTS) via increasing association of estrogen receptor beta (ERbeta) with Dishevelled2 (Dvl2) in AR non-responsive human PrCa DU145 cells. (Liu 2010). This analysis offers a new insight of the non-genomic effect of these steroid hormones. To validate the hypothesis based on data from prostate stromal and epithelial cell cultures, we are surveying frozen prostate cancer tissues with reactive stromal prostate characteristics for possible androgenic metabolites. We are working in collaboration with investigators in NCI and SAIC to analyze steroid metabolites in situ using liquid chromatography/ tandem mass spectrometry (LC/MS-MS), and in parallel, determine levels of markers for reactive stroma using digital image analysis of stained sections. We have examined the effect of TGF beta-1 on androstenedione and testosterone production in cultured prostate stromal cells as well as the enzymes that are involved in steroid metabolism including 3beta-HSD, 17betaHSD1, and 17betaHSD2, and 17betaHSD5. Inhibitors (siRNA) to hydroxysteroid dehydrogenase (HSD) enzymes blocked TGF beta-1 effects on DHEA metabolism and associations were found between HSDs and TGF-beta-1 receptors. TGF beta-1 also inhibited Phase II detoxifying pathways regulated by Nrf2, including the reduction of AKR1C1 enzymes which decrease available DHT. Additional work implies that phytoestrogens including red clover isoflavones, naringenins, and both ERalpha and ERbeta agonists can reverse epithelial PSA production in DHEA and TGF beta-1 treated cocultures. This in vitro model cannot provide data on all the complex physiological regulatory mechanisms inherent in prostate tissues. It is also difficult to extrapolate from human cell cultures, to clinical effects in humans. Nevertheless, DHEA-treated LAPC-4 and 6S cocultures provide a useful preclinical model to elaborate immune/hormone-mediated stromal cell signaling and to identify the mechanisms involved in metabolism of DHEA in prostate growth and gene regulation as well as intracrine and paracrine pathways and mediators of hormone and immunological action in human prostate cells. The stromal epithelial interface is an important target for various botanical agents or traditional medicines acting as therapeutic agents to normalize hormone metabolism or stromal regulation of epithelial cells. These CAM agents may have many different chemical constituents that are symbiotic and multi-mechanistic. This systems approach represents an experimental challenge in its anti-reductionist nature, yet addresses a vital need to understand the contributions of multiple disciplines in both cancer and CAM research.